ProjectSummary/Abstract Soil-transmitted helminth or soil-transmitted nematode (STN) infections are intestinal parasitic nematodes, mainlyAscaris,hookworms,andwhipworms.Theyareamongstthemostprevalentparasitesonearthand cause severe morbidity in children, including growth stunting, intellectual and educational impairment, malnutrition, anemia,andlowerfutureearning;?theyalsohavesignificantimpactsonpregnantwomenandworkerproductivity. Single dose mass drug administration (MDA) to treat STNs relies on a single drug class, the benzimidazoles (BZs). BZs have poor efficacy against whipworms and highly variable efficacy against hookworms. BZ resistance alleles have been detected in STNs and there are clear examples of low BZ efficacy against all parasites. New mechanism-of-action and broadly potent therapies for STNs are urgently needed. The soil- bacterium Bacillus thuringiensis (Bt) is the number one biological insecticide agent in the world. The insect- activecomponentsarecrystal(Cry)proteinsthatkillinsectsbutthatareharmlesstovertebrates(noeffect>1000 mg/kg). Insecticidal Bt Cry proteins are expressed in transgenicfood crops (e.g., >80% of USA corn) andare FDAapprovedforingestion.Thenematode-activeCryprotein,Cry5B(relatedtoinsecticidalCryproteins),cures hookwormandAscarisinfectionsinlargeanimalsandcankillwhipwormsinvitro.NovelCry5Bmicrobiologically- derivedformscalledIBaCCandPCC,whicharecompatiblewithMDA,havebeendeveloped.TheCryprotein, Cry21A,has also emerged asa potentially highly potent anthelminticagainstSTN parasites andan excellent stacking partner for Cry5B. Cry5B and Cry21A make a combination predicted to be difficult for nematodes to resist. This proposal aims to optimize Cry5B and Cry21A against all STNs by 1. taking advantage of the tremendousresourceofnaturalvariantsoftheseproteinsfoundintheenvironmentaspartofBt?sevolutionand survival,2.optimizingacidneutralizationandbacterialexpressionsystems,and3.testingkeyhypothesesabout howbest totarget whipworms, the most difficultof the parasites to treat. Moderate throughput in vitro assays will be used toefficiently inform in vivoassays for all threeparasite classes. Efficacy of Cry proteins against multidrug resistant hookworms will be studied. Potential resistance against Cry proteins will be addressed upfront by studying how Cry5B and Cry21A proteins can be combined to provide resistance-busting efficacy;? stackingofCryproteinsisnowthenormforinsecticidalapplication.Thisproposalisuniquein1.applyingasafe and validated natural resource, Bt, to a wholly new application, control of parasitic diseases of poverty;? 2. applyingmicrobiologytocontrolSTNs;?3.workingwithallclassesofSTNs;?and4.anticipatingresistanceissues upfront.TheaimattheendofthisstudyistohavedevelopedastackedCryproteintherapeutictoeffectasingle dosecureforhookworms,Ascaris,andwhipwormat?1mg/kgwhileaddressingresistanceissues.Futureplans includepediatricformulations,preclinicaltoxstudies,cGMPmanufacturing,andclinicaldevelopmenttoaddress theunmetneedtobroadlycureSTNinfectionsinsafely,sustainably,andeconomically.